Methods for liquid chromatography fluidic monitoring

ABSTRACT

A liquid chromatography monitoring system comprises a computer or electronic controller comprising computer-readable instructions operable to: (a) draw a fluid into a syringe pump; (b) configure a valve so as to fluidically couple the pump to either a fluidic pathway through a fluidic system or to a plug that prevents fluid flow; (c) cause the syringe pump to progressively compress the fluid therein or expel the fluid to the fluidic pathway, while measuring a pressure of the fluid; (d) determine a profile of the variation of the measured pressure; (e) compare the determined profile to an expected profile that depends upon the fluid; and (f) provide a notification of a sub-optimal operating condition or malfunction if the determined profile varies from the expected profile by greater than a predetermined tolerance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. application Ser. No.16/052,005, to be issued as U.S. Pat. No. 10,627,373, which is aDivisional of U.S. application Ser. No. 14/353,278, now U.S. Pat. No.10,054,569, which is the United States National Stage Application, under35 USC 371, of International Application No. PCT/US2012/034895 having aninternational filing date of Apr. 25, 2012 and designating the UnitedStates which is a Continuation-in-Part and claims the benefit of thefiling date, under 35 USC 365(c) and 35 USC 120, of InternationalApplication No. PCT/US2011/058230 having an international filing date ofOct. 28, 2011 and designating the United States which claims the benefitof the filing date, under 35 USC 119(e), of U.S. Provisional Application61/408,046 filed on Oct. 29, 2010, all said applications incorporated byreference herein in their entireties.

TECHNICAL FIELD

This invention generally relates to chromatography, and moreparticularly to an automated apparatus and method for monitoring thecorrectness of the installation and placement of solvents, mobile phasesor other reagents within a chromatograph instrument or system.

BACKGROUND ART

Liquid chromatography (LC) is well-known in the fields of chemicalseparation, compound purification and chemical analysis. A liquidchromatograph generally includes a separation column that comprises acapillary tube that is packed with a permeable solid material thateither is, itself, a chromatographic stationary phase or otherwisecomprises or supports a chromatographic stationary phase. A mobilephase, which is a fluid mixture comprising a compound of interest forpurification or separation as well as one or more solvents, is caused toflow through the column under pressure from an input end to an outputend. Generally, the chemical properties of the stationary phase and themobile phase solvents are such that the degree of partitioning of thecompound of interest between the mobile phase and the stationary phaseis different from the degree of partitioning of other compounds withinthe fluid. As a result, the degree of retention or time of retention ofthe compound of interest within the column is different from the degreeor time of retention of the other compounds, thus causing a physicalseparation or at least partial purification of the compound of interestfrom the other compounds.

There are numerous solvents available for liquid chromatography. Forinstance, the HPLC solvents available under the Fluka® brand name fromSigma-Aldrich Corporation (3050 Spruce Street, St. Louis, Mo. 63103 USA)include: water, Acetonitrile, Benzene, 1-Butanol, 2-Butoxyethanol,tert-Butyl methyl ether, Carbon tetrachloride, 1-Chlorobutane,Chloroform, 2-Chloropropane, Cyclohexane, Cyclopentane,1,2-Dichloroethane, Dichloromethane, Diethyl ether, 1,2-Dimethoxyethane,N,N-Dimethylacetamide, Dioxane, Ethanol, Ethanol, Ethyl acetate,Heptane, Hexane, Isooctane, Methanol, Methanol, Methyl acetate,Nitromethane, Pentane, 1-Propanol, 2-Propanol, 2-Propanol,Tetrachloroethylene, Tetrahydrofuran, and Toluene.

Within a chromatograph instrument or system, solvents or other reagentsare generally made available to the various columns, pumps, valves andassociated interconnecting tubing lines by means of a dedicated rack orcompartment. The rack or compartment generally comprises a dedicatedstorage area for the set of solvents or other reagents that willroutinely be needed or that may be needed by the chromatographinstrument or system during the course of several separations. Thereagent rack is generally designed to be accessed by an operator ortechnician at such times that one or more solvents or reagents need tobe replaced, having been depleted over the course of operation of theinstrument or system.

Successful chromatographic separations depend on specific chemicalinteractions of the various analytes and other components with astationary phase and with the various chemical constituents of a mobilephase. Because different analytes have different respective chemicalproperties, it is important that the correct set of solvents or reagentsfor an analysis at hand are mixed with a sample containing orpotentially containing any particular analyte. Therefore, the variousdifferent solvents or reagents are provided in respective dedicatedbottles or other containers within a reagent or solvent rack orcompartment. The different containers or bottles either have respectiveassigned locations within the rack or compartment or are associated withdifferent respective assigned draw tubes for aspiration of the solventor reagent into the system.

Because of the specificity of solvents or other reagents required forany particular chromatographic analysis protocol, it is important thatthese materials are not confused with one another (or with completelydifferent substances) or misplaced within a reagent or solvent rack orcompartment. Although reagents, solvents and other required chemicalsare generally supplied by manufacturers in well-labeled containers,these materials may be re-distributed into smaller containers within alaboratory environment. The smaller containers may be multi-purpose,initially-unlabeled vessels which require appropriate manual labelingupon initial receipt of material transferred from a manufacturer'soriginal container. The manual label applied in a laboratory may be anon-permanent label. After manual labeling, the small transfer vesselmay be handled within the laboratory many times and by many differentusers, since multiple replenishments from a large-volume manufacturer's“bulk” container may be required as the material within the vessel isroutinely consumed. The same vessel may be re-inserted into a solvent orreagent rack many times.

Many opportunities for operator error will occur over the course of themultiple handlings of the transfer vessel or, occasionally, even amanufacturer's original container. For instance, a temporary label maybe lost and replaced with an incorrect label. Even if the label iscorrect, the operator may transfer the wrong material into the transfervessel. Even if the label and material are correct, the operator maymis-place the vessel within a reagent rack or compartment. Conventionalchromatograph systems are designed to expect that particular solvents orreagents will be drawn into particular respective tubing lines. If anincorrect material is supplied, through any one or more of the errorslisted above, the chromatograph will continue to perform thepre-programmed steps of an analysis protocol with the wrong material.This may lead to incorrect or poor-quality results, necessitatingrepetition of many faulty analyses. In a worst-case scenario, the errormay never be discovered, and inappropriate actions may be taken based onthe incorrect analytical results. Nonetheless, by comparing theproperties of a solvent—such as viscosity and compressibility—with theexpected values which can be obtained through user input or by means ofa sensor mechanism, such as bar code, the solvent identity can bevalidated. Accordingly, there is a need in the art for an automatedchromatograph system that can take automated procedural steps in anattempt to recognize unexpected solvents or reagents before analysissteps are performed unexpected material and that can raise an operatoralert if any such errors are detected.

Liquid chromatography systems utilized in clinical laboratories or forpurposes of drug discovery may remain in near continuous operation overlong periods of time. As a result of wear, repeated handling, repeatedpressurization, multiple replacements of samples, etc., occasional orperiodic situations or conditions may occur which result in sub-optimalperformance of or even instrumental malfunctions in chromatographicsystems. For example, as a result of long term repeated pressurizationof fluid lines and other fluidic components, leaks may develop whicheither lead to undesirable loss of fluids from a fluidic system or,perhaps, undesirable ingestion of air into the system. Repeatedreplacement of sample vials or fluid or solvent containers may lead tocontamination of fluid lines by particulates or ingested air. Further,since many components such as pumps and valves undergo repeatedmechanical operation, long term wear of such components may occur which,if not addressed, may lead to loss of precision, loss or pressureintegrity or even total malfunction of one or more components. Finally,undesirable pressure imbalances may occur within fluidic systemscomprising various fluidic sub-systems, each sub-system having its ownrespective pumps. Accordingly, there are needs in the art for methodsfor monitoring the performance of chromatographic systems for thepurpose of detecting sub-optimal conditions, deterioration ofperformance, possible future failures, etc. and for warning users of theneed to take corrective action or notifying users of estimated remaininguseful lifetimes of components. Moreover, there is a need in the art foran automated chromatograph system that can perform such monitoring andprovide such warnings or notifications automatically. Preferably, liquidchromatograph (LC) system self-diagnostics and monitoring should includeself-diagnoses, validation and troubleshooting of i) the pump and ii)the LC system plumbing for leakage, air bubbles and fluid pathwayblocking.

There is also a need in the art for methods for balancing pressuresbetween different fluidic sub-systems. The compressibility of an LCsolvent affects the flow rate which in turn affects the chromatographicperformance. This effect is an issue for all high-performance (orhigh-pressure) liquid chromatography (HPLC) systems in general and forthose that use syringe pumps in particular. This effect is one of themain drawbacks associated with the syringe type of pump, although suchsyringe pumps provide other advantages such as smooth gradients and ahigh degree of robustness. M. Martein, et. al (“The use of syringe-typeof pumps in liquid chromatography in order to achieve a constantflow-rate”, Journal of Chromatography, 112, 1975) concluded that “[i]tis therefore not surprising that the syringe-type pumps have evolvedinto very sophisticated and expensive devices” in order to compensatethe compressibility issue. Even so “the use of syringe-type pumps isoften more difficult and less satisfactory than the use of other typesof pumps.”

DISCLOSURE OF INVENTION

The present disclosure addresses the above-noted needs in theconventional art through the teaching of methods and systems formonitoring properties of fluids provided to liquid chromatographysystems and comparing the monitored properties to the values that areexpected if correct fluids are provided. Such methods and systems arealso capable of monitoring the leak-tight worthiness of pumps and othermechanical or fluid-containing components of the liquid chromatographysystems.

The present teachings address the issue of the sensitivity ofsyringe-pump systems to liquid compressibility in at least two ways.First, a general-purpose compressibility compensation algorithm can beapplied. The algorithm can compare the compressibility of a solvent in acompressed volume within a specific time with the known compressibilityof the expected solvent. Then, an actual flow rate can be obtained totake account of the effect of the compressibility. The expected flowrate can be set as a target for the pump to achieve. This methodeliminates the need of the extra flow rate sensors. A controller such asPID (proportional-integral-derivative) can be used to achieve the targetflow rate.

Secondly, the various scenarios exhibiting the most serious effects ofcompressibility are addressed individually. Three different problematicscenarios are investigated: i). a pump undergoing connection to apressurized fluid pathway, ii) the achievement of flow rate withpressure and iii) situations in which different fluid pathway/subsystemspossibly having different pressures are interconnected or disconnectedfrom one another during the operation of a liquid chromatograph. In thefirst such scenario, the pressurized fluid pathway might flow back tothe unpressurized pump to compress the fluid inside the pump, therebycausing a sudden pressure drop and unintended solvent mixing. In thesecond scenario, the flow rate with pressure takes time to reach thepressure equilibrium and to reach the specified flow rate as a result ofthe compressibility of the solvent. The time taken to reach theequilibrium is determined by the compressibility of the solvent, thepressure, the flow rate and the solvent. The equilibration time couldrange from several seconds to more than hours. In the third scenario,the pressure imbalance could cause sudden unintended large fluid flowfrom a high-pressure subsystem to a low-pressurize sub-system. Even witha one-way fluid component such as check valve to prevent backflow, thelow-pressure subsystem needs time to reach pressure equilibrium with thehigh pressure subsystem. The consequent difference between the actualand expected flow rates as a result of the compressibility is so largeas to dramatically affect the performance of the liquid chromatograph.

By addressing each of the above scenarios individually, an optimizedsimple algorithm can be developed to only solve the problem associatedwith the particular scenario. This targeted approach can achieve thebest performance, in contrast to employing a generalized algorithm. If ageneralized method is used for all these scenarios, then the bestperformance is difficult to achieve and the method could be verycomplicated.

In accordance with a first aspect of the present teachings, there isdisclosed a system for providing a solvent or reagent to a liquidchromatography system comprising: a valve comprising a common port and aplurality of other ports, configurable such that the common port may befluidically coupled to any one of the other ports; a pump fluidicallycoupled to the common port of the valve; a plug configured to block flowthrough a first one of said other ports of the valve; a containercontaining the solvent or reagent, said container fluidically coupled toa second one of said other ports of the valve; and a pressure gauge orsensor configured to measure fluid pressure within the pump, wherein thesolvent or reagent is provided to the liquid chromatography system by afourth one of the other ports. The pumps may comprise syringe pumps. Thesystem may further comprise a fluid tubing line having a knownresistance to fluid flow fluidically coupled to a third one of saidother ports of the valve. Alternatively, the system may further comprisea fluid resistance such as a fluid tubing line, a column, etc., whereinthe fluid resistance could be either known or unknown as long as it isconstant. The system may further comprise a computer or electroniccontroller electrically or electronically coupled to the pump, thepressure gauge or sensor and the valve. The system may still furthercomprise an electronically-readable medium having thereon programinstructions readable by the computer or electronic controller, saidinstructions operable to cause the computer or electronic controller tomeasure readings of the pressure gauge or sensor while causing the pumpto apply a force to a fluid therein so as to urge said fluid to eitherthe first or third port of the valve.

In accordance with a second aspect of the present teachings, there isdisclosed a method for monitoring fluids within a liquid chromatographysystem comprising: (a) configuring a valve so as to draw a fluid from acontainer into a pump; (b) configuring the valve so as to fluidicallycouple the pump to a port of the valve that is coupled to a plug thatprevents fluid flow through said port; (c) causing the pump toprogressively compress the fluid therein, while measuring a pressure ofthe fluid in the pump; and (d) determining if a rate of increase of themeasured pressure substantially matches an expected value. Additionalsteps of the method may comprise one or more of: (e) upon measuring amaximum pressure or, alternatively, any suitable pre-determinedpressure, maintaining a piston of the pump in a constant position for atime of pre-determined length while continuing to measure the pressureof the fluid in the pump; and (f) determining if a decrease of themeasured pressure by more than an acceptable value occurred during thetime period. Still further steps of the method may comprise: (g) causingthe pump to relieve the pressure of the fluid in the pump; (h)configuring the valve so as to fluidically couple the pump to a fluidpathway having a pre-determined resistance to fluid flow therethrough;(i) causing the pump to displace fluid into fluid pathway at a set flowrate while measuring the pressure of the fluid in the pump; and (j)determining if an increase of the measured pressure during the fluiddisplacement substantially matches a second expected value. The methodmay include raising an alarm either that the fluid in the container maynot match expectations or that the pump may not be leak-free accordingto expectations depending on the measured increases or decreases in thepressure of the fluid in the pump.

In accordance with a third aspect of the present teachings, there isdisclosed a liquid chromatography system comprising: (a) a valve systemor fluid selecting apparatus having an output port and a plurality ofinput ports thereof, each of the plurality of input ports fluidicallycoupled to a respective fluid-providing sub-system, each fluid-providingsub-system comprising: (i) a valve comprising a common port and aplurality of other ports, configurable such that the common port may befluidically coupled to any one of the other ports; (ii) a pumpfluidically coupled to the common port of the valve; (iii) a plugconfigured to block flow through a first one of said other ports of thevalve; (iv) a container containing a fluid, said container fluidicallycoupled to a second one of said other ports of the valve; (v) a pressuregauge or sensor configured to measure fluid pressure within the pump;and (vi) a third one of the other ports fluidically coupled to the valvesystem or fluid selecting apparatus; (b) a chromatograph column having afirst end fluidically coupled to the output of the valve system or fluidselecting apparatus and a second end; and (c) a detector fluidicallycoupled to the second end of the chromatograph column. At least onefluid-providing sub-system may further comprise (vii) a fluid tubingline having a known resistance to fluid flow fluidically coupled to afourth one of the other ports of the valve of the respectivefluid-providing sub-system.

In accordance with a fourth aspect of the present teachings, there isdisclosed a method for monitoring a fluidic system of a liquidchromatography system, wherein the system comprises a valve, a containerhaving a known fluid therein and a syringe pump having a piston, andwherein the syringe pump is fluidically coupled to the valve. Accordingto this aspect, the method is characterized by: (a) drawing the fluidfrom the container into the syringe pump; (b) configuring the valve soas to fluidically couple the pump to a port of the valve that is coupledto either a fluidic pathway through the fluidic system or to a plug thatprevents fluid flow through said port; (c) causing the piston of thesyringe pump to move at a predetermined rate in a direction so as toprogressively compress the fluid therein or expel the fluid to thefluidic pathway, while measuring a pressure of the fluid; (d)determining a profile of the variation of the measured pressure for thetime that the piston is caused to move; (e) comparing the determinedprofile to an expected profile that depends upon the fluid; and (f)providing a notification of a sub-optimal operating condition ormalfunction if the determined profile varies from the expected profileby greater than a predetermined tolerance. In some instances, anexpected profile may be reduced to simply one or more characteristicrates of change of pressure, such as a rate of pressure increase or arate of pressure decrease.

In various embodiments, the step (b) of configuring the valve maycomprise configuring the valve so as to fluidically couple the pump to aport of the valve that is coupled to a fluidic pathway having anintentional flow blockage therein that prevents flow through the fluidicsystem beyond the intentional flow blockage. Such an intentional flowblockage may comprise one or more closed valves or may be provided at anominal position of a chromatographic column within the fluidic system.In some embodiments, the intentional flow blockage is provided in acartridge that is interchangeable with and that is disposed within thefluidic system at the nominal position of a two-column-bearingcartridge.

In various embodiments, the step (b) comprises configuring the valve soas to fluidically couple the pump to a port of the valve that is coupledto a fluidic pathway that includes a length of empty tubing thatreplaces a chromatographic column at the nominal column position and thestep (f) comprises providing a notification that the fluid pathway isblocked if the determined pressure profile includes a pressure increasethat exceeds an expected increase in pressure by greater than thepredetermined tolerance. In various other embodiments, the step (b) ofconfiguring the valve comprises configuring the valve so as tofluidically couple the pump to a port of the valve that is coupled to afluidic pathway having a known resistance to fluid flow, and the step(e) of comparing the determined profile to an expected profile comprisescomparing the determined profile to an oscillatory profile, theoscillations of said profile relating to mechanical movement within thesyringe pump.

In various embodiments, the step (f) of providing a notification maycomprise providing a notification that an air or gas bubble is presentwithin the fluidic system if the determined pressure profile includes adelay in an increase in pressure, relative to the expected profile.Various embodiments of the method may include the additional steps of(g) causing the piston of the syringe pump to remain in a fixedposition, while measuring a pressure of the fluid; (h) determining arate of decrease of the measured pressure while the piston is in thefixed position; (i) comparing the determined rate of pressure decreaseto a model relating rate of pressure decrease to remaining pumplifetime; and (j) either providing a prediction of remaining pumplifetime or providing a warning that the pump should be replaced orserviced based on the comparing.

In accordance with a fifth aspect of the present teachings, there isdisclosed a method of balancing fluid pressure between a first portionand a second portion of a fluidic system of a liquid chromatographysystem, wherein the second portion is initially at higher fluid pressurethan the second portion, wherein the liquid chromatography systemcomprises a coupling system that may either fluidically interconnect ormutually isolate the first and second fluidic system portions, andwherein the liquid chromatography system further comprises a firstsyringe pump, a selection valve that is fluidically coupled to the firstsyringe pump and to the first portion of the fluidic system, a secondsyringe pump that is fluidically coupled to the second portion of thefluidic system, a first pressure sensor configured to measure pressurewithin the first syringe pump and a second pressure sensor configured tomeasure pressure within the fluidic system. According to this aspect,the method is characterized by: (a) configuring the selection valve soas to fluidically couple the first syringe pump to a port of the valvethat is coupled to a plug that prevents fluid flow through said port;(b) compressing a fluid within the first syringe pump so that a readingof the first pressure sensor matches a reading of the second pressuresensor; (c) configuring the selection valve so as to fluidically couplethe first syringe pump to a port of the valve that is coupled to thefirst portion of the fluidic system; and (d) fluidically interconnectingthe first and second portions of the fluidic system using the couplingsystem.

In various embodiments, the first portion of the fluidic system includesa first chromatographic column and the second portion of the fluidicsystem includes a second chromatographic column. The coupling system maycomprise a one-way check valve disposed within the first portion of thefluidic system and a mixing tee coupler at which fluids from the firstand second portions are mixed. Alternatively, the fluid coupling maycomprise the one-way check and a multiple-port rotary valve, whereinfluids from the first and second portions may be mixed. In someembodiments, the first portion of the fluidic system comprises achromatography column-loading sub-system and the second portion of thefluidic system comprises an eluting sub-system.

In accordance with another aspect of the present teachings, there isdisclosed a method of operating a liquid chromatography system, whereinthe system includes a chromatographic column having a nominal operatingpressure, a syringe pump, a pressure sensor configured to measurepressure within the syringe pump, and a selection valve that isfluidically coupled between the syringe pump and the chromatographiccolumn. According to this aspect, the method is characterized by: (a)configuring the selection valve so as to fluidically couple the firstsyringe pump to a port of the valve that is coupled to a plug thatprevents fluid flow through said port; (b) compressing a fluid withinthe syringe pump until a reading of the pressure sensor matches thenominal operating pressure; (c) configuring the selection valve so as tofluidically couple the syringe pump to a port of the valve that iscoupled to the chromatographic column; and (d) operating the syringepump so as to pump a sample fluid through the chromatographic column.

In accordance with yet another aspect of the present teachings, there isdisclosed a method for monitoring for the existence of leaks within afluidic system of a liquid chromatography system, wherein the liquidchromatography system comprises a valve, a container having a knownfluid therein, a syringe pump having a piston, and a pressure sensorwherein the syringe pump is fluidically coupled to the valve. Accordingto this aspect, the method is characterized by: (a) drawing the fluidfrom the container into the syringe pump; (b) configuring the valve soas to fluidically couple the pump to a port of the valve that is coupledto either a fluidic pathway of the fluidic system having an intentionalflow blockage therein or to a plug that prevents fluid flow through saidport; (c) causing the piston of the syringe pump to move at apredetermined rate in a direction so as to increase the pressure of thefluid therein or within the fluidic pathway; (d) causing the piston ofthe syringe pump to remain in a fixed position, while measuring apressure of the fluid; (e) determining a rate of decrease of themeasured pressure while the piston is in the fixed position; and (f)providing a warning that a leak is present if the determined rate ofpressure decrease exceeds a pre-determined threshold value. In someembodiments, the intentional flow blockage may be provided at a nominalposition of a chromatographic column within the fluidic system. In someembodiments, the intentional flow blockage is provided in a cartridgethat is interchangeable with and that is disposed within the fluidicsystem at the nominal position of a two-column-bearing cartridge. Invarious embodiments in which the valve is configured to fluidicallycouple the pump to the plug, the determined rate of pressure decreasemay be compared to a model relating rate of pressure decrease toremaining pump lifetime; and a prediction of remaining pump lifetime maybe made based on the comparing.

BRIEF DESCRIPTION OF DRAWINGS

The above noted and various other aspects of the present invention willbecome apparent from the following description which is given by way ofexample only and with reference to the accompanying drawings, not drawnto scale, in which:

FIG. 1 is a schematic illustration of a generalized conventional liquidchromatography-mass (LCMS) spectrometry system;

FIG. 2 is a schematic illustration of an LCMS system in accordance withthe present teachings;

FIG. 3 is an illustration of an exemplary rotary valve assembly as maybe employed within an apparatus in accordance with the presentteachings;

FIG. 4A is a schematic diagram of an exemplary two column LCMS systememploying a fluid monitoring portion in accordance with the presentteachings;

FIG. 4B is a schematic diagram of a second exemplary two column LCMSsystem employing a fluid monitoring portion in accordance with thepresent teachings;

FIG. 5 is a schematic diagram showing an example of valve configurationsand fluid flow paths steps that may be employed in a chromatographymethod employing the system of FIG. 4A;

FIG. 6 is a flow diagram of a method for chromatography fluid monitoringand verification in accordance with the present teachings;

FIG. 7A is a schematic illustration of a sample source portion of anLCMS system according to some embodiments in accordance with the presentteachings;

FIG. 7B is a schematic illustration a solvent source portion of stillanother LCMS system according to some embodiments in accordance with thepresent teachings;

FIG. 7C is a schematic illustration a solvent source portion of yetanother LCMS system according to some embodiments in accordance with thepresent teachings;

FIG. 8 is a graph showing a set of plots of observed pressure versustime for respective fluids as the fluids are compressed against aplugged port by movement of a syringe pump piston;

FIG. 9A is a schematic graph illustrating, in a general sense, apressure profile within a syringe pump versus time in a situation inwhich the syringe pump is used to compress a fluid against a pluggedport and subsequently maintain pressure;

FIG. 9B is a graph showing experimental data of the observed pressureleak rate for a syringe pump (with valve) versus the number of cycles ofactuation;

FIG. 10 is a graph showing two plots of observed pump chamber pressureversus time as a fluid is pumped from a syringe pump into a resistivetubing at a flow rate of 200 μl per minute;

FIGS. 11A-11D are illustrations, respectively, of a normalchromatographic cartridge having two columns, a cartridge for systemtest purposes having two unobstructed tubes, a second cartridge forsystem test purposes having one unobstructed tube and one plugged line,and a third cartridge for system test purposes having two plugged lines;

FIG. 12A is a flow diagram of a method for chromatographic system leakmonitoring and detection in accordance with the present teachings;

FIG. 12B is a flow diagram of a second method for chromatographic systemleak monitoring and detection in accordance with the present teachings;

FIG. 13 is a flow diagram of a method for chromatography pump lifetimemonitoring and testing in accordance with the present teachings;

FIG. 14 is a flow diagram of a method for monitoring a liquidchromatography system in accordance with the present teachings;

FIG. 15 is a flow diagram of another method for monitoring a liquidchromatography system in accordance with the present teachings;

FIG. 16A is a flow diagram of a method for balancing pressure indifferent portions of a liquid chromatography fluidic system inaccordance with the present teachings;

FIG. 16B is a flow diagram of a method for compensating pressuredifferences in different portions of a liquid chromatography fluidicsystem in accordance with the present teachings; and

FIG. 17 is a flow diagram of a general method in accordance with thepresent teachings for performing multiple fluidic identification andpump diagnostic functions.

MODES FOR CARRYING OUT THE INVENTION

The following description is presented to enable any person skilled inthe art to make and use the invention, and is provided in the context ofa particular application and its requirements. Various modifications tothe described embodiments will be readily apparent to those skilled inthe art and the generic principles herein may be applied to otherembodiments. Thus, the present invention is not intended to be limitedto the embodiments and examples shown but is to be accorded the widestpossible scope in accordance with the features and principles shown anddescribed. To appreciate the features of the present invention ingreater detail, please refer to FIGS. 1-17 in conjunction with thefollowing discussion.

FIG. 1 is a schematic illustration of a conventional liquidchromatography (LC) system. The system 10 shown in FIG. 1 comprises achromatograph column 7 for separating a liquid chemical mixture into itsconstituent substances and a detector 20 (such as spectrophotometer or amass spectrometer) fluidically coupled to the column 7 for detecting oridentifying some or all of the separated constituent substances as theyare received, in sequence from the column 7. The column receives a fluidstream comprising one or more selected solvent fluids or reagentssupplied from containers 8 as well as a sample of interest from samplesource 4. The various different solvent or reagent fluids, which maycomprise a chromatographic mobile phase, are delivered along fluidtubing lines 6 a to valve or fluid selecting or mixing apparatus 9 whichmay mix the fluids or select a particular fluid. As illustrated, theapparatus 9 is a three-way valve but may comprise a more complex valveor valve system if more than two different solvent fluids are provided.Alternatively, the apparatus 9 could comprise a simple mixing junctionor mixing chamber.

The fluids are drawn into the system 10 and propelled to thechromatographic column 7 therein by means of a pump 11 that isfluidically coupled to the output of the valve or fluid selectingapparatus 9 by fluid tubing line 6 d. Alternatively, the singleillustrated fluid pump 11 could be replaced separate pumps—one for eachsolvent or reagent—disposed at positions 11 a in fluid tubing lines 6 a.The fluids output from the pump or pumps are delivered to a sampleinjector apparatus 5 along fluid tubing line 6 e and are mixed togetherwith a sample provided from the sample source 4. The sample injectorapparatus 5 may comprise, in a well-known fashion, a multiple-portrotary valve 23 and an injection loop 6 p fluidically coupled betweentwo of the ports.

An input of the column 7 of system 10 is fluidically coupled to andreceives a mixture of sample and solvent fluids from an output port ofthe sample injector apparatus 5 by fluid tubing line 6 f. Differentialpartitioning of the various chemical constituents of the mixture betweenthe mobile phase and a stationary phase packed within the column leadsto differential retention of the various constituents within the columnand consequent different respective times of elution of the constituentsfrom the column output to fluid tubing line 6 g. An optional valve 12may separate the eluting substances, either continuously or at varioustimes, into a portion that is delivered to waste container 14 alongfluid tubing line 6 w and an analysis portion that is delivered thedetector 20 along fluid tubing line 6 h.

The conventional system 10 shown in FIG. 1 is susceptible to thepossible handling errors of a transfer vessel or, occasionally, even amanufacturer's original container, as described supra herein.Accordingly, FIG. 2 provides a schematic illustration of an improvedLCMS system, system 50, in accordance with the present teachings. In thesystem 50, the solvents or reagents that are drawn from containers 8into the fluid tubing lines 6 a are delivered to a multiple-port sourcevalve 9 r.

As indicated in the inset 33 of FIG. 2, the source valve 9 r comprises acommon port p0 as well as several dedicated ports—in the illustratedsystem, six dedicated ports denoted as ports p1-p6. The source valve 9 rmay comprise a type of a known multiple-port rotary valve, such as thevalves known as Rheodyne valves sold by IDEX Health & Science, 619 OakStreet Oak Harbor, Wash. USA. A more-detailed illustration of anexemplary source valve 9 r is shown in FIG. 3. The source valve 9 rshown in FIG. 3 comprises, in known fashion, a stator portion 66 havinga plurality of fluid passages 68 therein passing through the statorportion from one end to another end and a rotor portion 62 having agroove or channel 64 on a side facing the stator portion. As shown, thestator comprises a central fluid passage (corresponding to the port p0shown in FIG. 2) as well as six peripheral fluid passages (correspondingto the ports p1-p6 shown in FIG. 2) radially disposed about the centralpassage. The rotor portion 62 may rotate, as illustrated by thedouble-headed arrow in FIG. 3, so that any one of the peripheralpassages may be fluidically connected to the central passage, within thesource valve 9 r, by means of the groove or channel 64. Although arotary valve is shown, the invention is not intended to be limited tosuch, as the rotary valve is but one example of a valve which may beemployed.

Returning to FIG. 2, the solvents or reagents are drawn from theirrespective containers 8 into and through the source valve 9 r by meansof a syringe pump 11 s which is coupled to the central or common port p0as well as to a pressure sensor or gauge 16. Two different solvent orreagent fluids may be fluidically coupled to two respective ports, forexample, ports p5 and p6, of the source valve 9 r by means of the fluidtubing lines 6 a. To draw either one of these fluids into the cylinderof the syringe pump, the source valve is configured such that thecentral port p0 is fluidically coupled to one of ports p5 and p6 while apiston of the syringe pump is withdrawn.

A computer or other electronic logic controller 32 may be includedwithin the system 50 so as to receive information from and transmitcontrol signals to various components of the system. The computer orother electronic logic controller 32 may be electronically coupled tothe pump 11 s, the pressure sensor or gauge 16 and the source valve 9 rby means of electronic communication lines 34 a, 34 b and 34 c,respectively. The computer or other electronic logic controller 32 mayalso be electronically coupled to other components of the system 50,although such couplings are not explicitly illustrated in FIG. 2.

One port, for example, port p1 of the source valve is blocked or pluggedso that fluid cannot exit through this port and may be a defaultposition of the source valve when the pump is not in use. If the sourcevalve is configured to dispense solvent to port p1 and force is appliedto the syringe pump piston, the pressure measured by sensor or gauge 16is expected to rise rapidly. In the absence of a leak, the rate ofpressure rise depends on fluid compressibility. The rate of pressurerise may be used to verify the correctness of a particular solvent orreagent, from among a limited number of choices. The use of the plugposition p1 as a source valve “output” can also be used to verify pumpseal performance and pump priming. Using a calibration fluid of knowncompressibility, a failure of the pressure to increase as expected or anunexpected pressure decrease can indicate an apparatus defect.

Another port, for instance, port p2 of the source valve 9 r is theoutput to the sample injector apparatus 5 via fluid tubing line 6 d.Another port, for instance, port p3 is used to output a small portion ofa previously aspirated solvent into a waste container through acalibrated length of resistive tubing 6 c. The tubing 6 c may comprise arestricted-diameter inner bore which provides a known resistance tofluid flow. If the source valve is configured to dispense solvent toport p3 and force is applied to the syringe pump piston, the solventwill be dispensed to the waste container 14 concurrent with a rise inpressure, as measured by sensor or gauge 16, that corresponds to solventviscosity. This measured pressure rise may be used to verify solventidentity, from among a limited number of choices. The relation betweenpressure rise and viscosity may be calibrated by dispensing acalibration fluid having known viscosity through port p3. Finally,another port, such as port p4, is an output to the waste container 14,using least resistance (e.g. regular) fluid tubing line 6 b, which isused for pump prime and purge operations.

With regards to the system 50 shown in FIG. 2, it is to be kept in mindthat the system may be expanded by including additional instances of thesub-set of components comprising: the one or more solvent or reagentcontainers 8, the source valve 9 r having a plugged port, the pump 11 s,the pressure sensor or gauge 16, the known-resistance tubing 6 c, thewaste container 14 and the other connecting fluid tubing lines 6 a, 6 band 6 d. This sub-set of components may be considered to comprise afluid-providing sub-system of the liquid chromatograph system 50.Alternatively or in addition, different respective solvents may beprovided in different respective instances of said fluid-providingsub-system, especially if a large number of solvents or reagents areprovided within the system.

Many liquid chromatography systems employ more than one chromatographiccolumn during fractionation, separation or purification of an analyte.For instance a first column may comprise a sample “cleanup” column and asecond column may comprise an analytical column. The cleanup column,according to some embodiments, may be a size exclusion or affinityliquid chromatography column or a High-Turbulence Liquid Chromatographycolumn used for matrix interference removal. For instance, a test samplemay applied to a first column (e.g., a clean-up column such as a CycloneP column or the like) at the inlet port, eluted with a solvent orsolvent mixture onto a second column (e.g., an analytical column such asa Hypersil Gold PFP or the like), and eluted with a solvent or solventmixture from the second column to the outlet port. Different solventmodes may be selected for eluting the analytes. For example, liquidchromatography may be performed using a gradient mode, an isocraticmode, or a polytyptic (i.e. mixed) mode.

FIGS. 4A and 4B are schematic diagrams of two exemplary two-column LCMSsystems employing a fluid monitoring portion in accordance with thepresent teachings. Sample injector 5 and fluid tubing line 6 f arereproduced from FIG. 2. For clarity of presentation, components upstreamof the sample injector (including the solvent or reagent containers 8,the source valve 9 r, the pump 11 s, the pressure sensor or gauge 16,the known-resistance tubing 6 c, the waste container 14, the connectingfluid tubing lines 6 a, 6 b and 6 d and the electronic communicationlines 34 a, 34 b and 34 c) are not shown but are considered to bepresent in the systems shown in FIGS. 4A-4B. Alternatively, the sampleinjector 5 shown in FIGS. 4A-4B may be replaced by a more-complex samplesource sub-system—possibly comprising multiple sample injectors, pumps,valves, mixing tee joints, solvent or reagent containers, and tubinglines. In both the system 70 (FIG. 4A) and the system 75 (FIG. 4B), twochromatograph columns—a first column 7 a and a second column 7 b—areutilized. The first column 7 a may advantageously comprise a cleanupcolumn that may be employed to separate certain classes or sub-sets ofcompounds from one another (e.g. large molecule versus small molecule,or polar versus non polar) with the fraction that may contain possibleanalyte substances retained and the other fraction discarded (or viceversa). The second column, column 7 b, is an analytical column that maybe similar to the single column 7 of the system 50 (FIG. 2). Theretained fraction eluted from the first, cleanup column 7 a may beseparated into particular isolated compounds by the second column 7 b.The eluted constituents may be provided to detector 20 along fluidtubing line 6 h.

As an example of a two-stage chromatographic separation, a TurboFlow®column (also known as a High Turbulance Liquid Chromatography or HTLCcolumn) may be employed as the cleanup column 7 a in a first separationstep in order to isolate and possibly concentrate a subset of compoundsbased on their size range or molecular weight range (or some otherproperty). TurboFlow® methods and apparatus are described in detail inU.S. Pat. Nos. 5,772,874; 5,919,368 and 6,149,816, all of which arehereby incorporated by reference in their entirety as if fully set forthherein. Briefly stated, the TurboFlow® apparatus and methods include orrelate to a chromatography column or body that is formed as asubstantially uniformly distributed multiplicity of rigid, solid, porousparticles having substantially uniform mean cross-section dimensions ordiameters of not less than about 30 μm, typically 50 μm or greater upto, but not limited to, 1000 μm in certain instances. The particles areselected from a range of various sizes and shapes and are held togetherin a body or column as by pressure, sintering and the like so thatinterstitial channels having a total interstitial volume of not lessthan about 45% of the total volume of the column are formed between theparticles. The surfaces of the particles, including the inner surfacesof the pores in the particles, are chromatographically active, as bybeing coated with chromatographic stationary phase layers.

Because of the nature of the particles and packing in a TurboFlow®column, the flow of the fluid mixture through the column can be at ahigh flow rate and is believed that, under such conditions, turbulentflow of the mixture is induced within at least a major portion of theinterstitial volume, and it is postulated that such turbulent flow infact enhances the rate of mass transfer, thus increasing the dynamiccapacity of the column From the principles of turbulence, diffusion, andchemistry, small sample molecules may be separated from a sample matrixin a TurboFlow® column Since small molecular weight molecules diffusefaster than large molecular weight molecules, the small sample compoundsdiffuse into the particle pores. The turbulent flow of the mobile phasequickly flushes the large sample compounds through the column to wastebefore they have an opportunity to diffuse into the particle pores. Ofthe sample molecules that enter the pores, those that have an affinityto the chemistry inside the pores bind to the internal surface of thecolumn particles. The small sample molecules that have a lower bindingaffinity quickly diffuse out of the pores and are flushed to waste. Achange in mobile phase, temperature or other parameter may then causethose molecules that were bound by the TurboFlow® column to elute to theanalytical column for further separation.

The flow of analyte bearing or other fluids—including samples, solventsand mixtures thereof possibly together with other chemicalcomponents—through the two chromatograph columns 7 a, 7 b of either thesystem 70 or the system 75 is controlled by two multi-port valves v1,v2, illustrated as valve system 45. Each valve may be a rotary valve ofa known type, such as Rheodyne valves in which a rotor portion comprisestwo or three channels that may fluidically interconnect various pairs ofadjacent ports, depending on the orientation of the rotor portion. Therotation and channels are schematically indicated, respectively, by adouble-headed arrow and by a set of dotted straight lines in each of thevalves v1 and v2. The first valve v1 may be configured to as tofluidically interconnect the members of three different pairs ofadjacent ports; the second valve may be configured so as to fluidicallyinterconnect a first pair of adjacent ports as well as all the ports ofa triplet of ports, as shown by the dotted line.

One port of the first valve v1 receives a fluid from fluid tubing line 6f. Fluid tubing lines 6 j and 6 k fluidically connect the ports of thefirst column 7 a to respective ports of the first valve; fluid tubingline 6 n fluidically connects a port of the first valve v1 to a port ofthe second valve v2 and another fluid tubing line 6 m fluidicallyinterconnects two ports of the first valve. The first valve v1 andassociated fluid tubing lines may be configured (as shown) such thatfluid may be caused to flow through the first column 7 a in eitherdirection.

In either the system 70 or the system 75, a port of the second valvereceives a fluid, possibly comprising various solvents or other chemicalconstituents or mixtures thereof, from a solvent source 3 via fluidtubing line 6 s. The solvent source 3 may comprise a sub-systemincluding various reagent containers as well as one or more syringepumps, rotary source valves, pressure sensors, resistive fluid tubinglines, mixing tee joints, waste containers and other interconnectingfluid tubing lines similar to corresponding features illustrated in FIG.2. Thus, the solvent source 3 may include a second parallel instance ofa fluid-providing sub-system as described with reference to the system50 or may comprise a more-complex fluid-providing sub-system. One ormore electronic communication lines 34 f may electronically couplevarious of the components of the solvent source 3 to the computer orother electronic logic controller 32 in similar fashion as shown in FIG.2.

Two ports of the second valve v2 may be plugged or otherwise unused, asindicated by hatch marks in FIGS. 4A, 4B. Another port of valve v2directs non-analyzed fluids to a waste container 14 along fluid tubingline 6 w. A final port of the valve v2 is fluidically coupled to aninlet port of the second chromatograph column 7 b via fluid tubing line6 q. One or more electronic communication lines 34 g may couple thecomputer or other electronic logic controller 32 to the valves v1, v2 soas to control their operation.

The system 75 illustrated in FIG. 4B is similar to the system 70illustrated in FIG. 4A with the exception that the two chromatographcolumns 7 a, 7 b are housed together in a cartridge or housing 40. Anexample of a two-column cartridge that may be employed as the cartridge40 is disclosed in a co-pending International (PCT) Application filed onOct. 28, 2011 titled “Modular Multiple-Column Chromatography Cartridge”(Attorney Docket No. 5854WO1/PCT; International Application No.PCT/US11/58229) and assigned to the assignee of the present inventionand incorporated herein by reference in its entirety. The cartridge 40may include, in addition to a housing, a computer-readableidentification (an indicator or identifier), such as a barcode or anRFID tag and may also include on-board computer readable memory, such asflash memory or any other form of electronic memory device, as well asan on-board electronic processor. The on-board memory, if present, maybe used to store data relating to the use of the columns of thecartridge, such as supported chromatographic methods or column usagehistory. Further, the cartridge 40 may include one or more heaters tomaintain one or the other of the columns at a temperature correspondingto an analysis protocol, as well as one or more sensors of temperatureor some other physical quantity. Accordingly, the cartridge may beelectronically coupled to the computer or other electronic logiccontroller 32 by an electronic communication line 34 h so as to, forinstance, transfer identification or other data to or from a memory unitof the cartridge, to control heaters or to monitor the sensors.

FIG. 5 is a schematic diagram showing an example of valve configurationsof the valve system 45 and fluid flow paths steps that may be employedin a chromatography method employing the system of FIG. 4A. The top,middle and bottom diagrams of FIG. 5 respectively illustrate: a sampleloading step in which an analyte-bearing fluid is delivered to the firstcolumn 7 a; a transfer step in which the at least partially purifiedanalyte is mixed with a solvent in valve v2 and transferred to thesecond column 7 b; and an eluting step, in which the analyte is elutedfrom the second column 7 b and transferred to the detector (not shown inFIG. 5). In these diagrams, different flow paths are distinguished fromone another by lines of different appearance (i.e., solid, dotted,dashed and dash-dot lines). Other modes of operation, utilizingalternative sets of valve configurations or sequences, are alsopossible.

Example 1—Fluid Monitoring and Verification

In accordance with the discussion presented above, FIG. 6 provides aflow diagram of a method 100 for chromatography fluid monitoring andverification in accordance with the present teachings. The method 100may be executed by software or firmware of the computer or otherelectronic logic controller 32 in conjunction with signals transmittedalong the electronic communication lines 34 a, 34 b and 34 c. In thefirst step, Step 102 of the method 100, a user selects an LC methodwhich includes solvents or reagents for which compressibility andviscosity information is available. In the next step, Step 104, a valve,such as the source valve 9 r, is selected such that a pump (forinstance, the syringe pump 11 s) draws a fluid from a designated solventor reagent bottle. In Step 106, the valve is configured so as tofluidically couple the pump cylinder, filled with the fluid drawn instep 104, to a plugged location, such as location p1 of the source valve9 r. In Step 108, the pump is operated so as to compress the fluidtherein, while monitoring pump pressure, such as with sensor or gauge16. Then, in the decision step, Step 110, if the observed rate ofpressure increase (from Step 108) does not substantially match anexpected value for an expected fluid, then the method execution iscaused to branch to an execution termination or interruption step, Step112, in which an alarm is raised that the solvent in the location fromwhich the fluid was drawn may not match expectations. Upon observing thealarm, a user may perform any appropriate tests or checks to determineif the correct solvent or reagent is loaded in the position from whichfluid was drawn. Depending upon the results of such tests or checks, theuser may replace the solvent or reagent and re-start execution of themethod 100 or, alternatively, may re-set the alarm (possibly, aftersolvent or reagent replacement) and continue execution of the methodfrom the interrupted point.

If, during execution of step 108 of the method 100 (FIG. 6), theobserved rate of pressure increase does indeed substantially match anexpected value for an expected fluid, then step 110 branches executionto Step 114 in which, after obtaining a maximum useable pressure, thepump piston is held in place for a set amount of time while continuingto measuring any change or changes in pressure. In the subsequentdecision step, Step 116, if an observed pressure drop is greater than anacceptable amount during the selected time, then execution is caused tobranch to an execution termination or interruption step, Step 118, inwhich an alarm is raised that the pump in the given location may not beleak-free in accordance with expectations. Upon observing the alarm, auser may perform any appropriate tests or checks to determine if thepump is operating correctly. Depending upon the results of such tests orchecks, the user may need to replace or repair the pump or othercomponents and re-start execution of the method 100 or, alternatively,may re-set the alarm and continue execution of the method from theinterrupted point.

The method 100 (FIG. 6) branches to Step 120 if, in Step 116, theobserved pressure drop is determined to not exceed the acceptable amountduring the selected time. In step 120, the pump may be operated, such asby moving a pump piston, so as to relieve the pressure in pump chamber.In the subsequent Step 122, the valve, such as source valve 9 r, isoperated so as to fluidically connect the pump with a fluid pathwayhaving a known fluid resistance; in Step 124, the pump is operated so asto inject the fluid into such pathway at a set flow rate while the pumppressure is measured. In the subsequent decision step, Step 126, if theobserved pressure increase (of Step 124) does not match an expectedvalue, then execution is caused to branch to a termination orinterruption step, Step 128, in which an alarm is raised that that thesolvent in the location from which the fluid was drawn may not matchexpectations. Upon observing the alarm, a user may perform anyappropriate tests or checks to determine if the correct solvent orreagent is loaded in the position from which fluid was drawn. Dependingupon the results of such tests or checks, the user may replace thesolvent or reagent and re-start execution of the method 100 or,alternatively, may re-set the alarm (possibly, after solvent or reagentreplacement) and continue execution of the method from the interruptedpoint.

Execution of the method 100 proceeds to Step 130 if all pressuremonitoring tests have yielded acceptable results. At this point, it maybe reported to a user that the pump and solvent check passed withacceptable measurements. Subsequently, the valve may be configured so asto dispense the solvent or reagent into the system along fluid tubingline 6 f (if the solvent is to be utilized) or to waste.

Alternative Hardware Configurations

FIG. 7A is a schematic illustration of a sample source sub-system of anLCMS system according to some embodiments in accordance with the presentteachings. The sub-system 200 shown in FIG. 7A may be employed as aloading system for loading analytes onto a chromatographic column. Thesub-system 200 comprises a sample injector apparatus 5 and may comprisetwo or more syringe pumps 11 s, respective associated pressure sensorsor gauges 16, respective associated selection valves 9 r, and respectivesolvent sources 8. Such components may be essentially similar tosimilarly-labeled components in FIG. 2. A dedicated waste container 14may be included as part of the sample-source sub-system 200 or,alternatively, a single waste container or drain manifold may beemployed for a chromatography system of which the sub-system 200 is apart.

Corresponding fluid tubing lines 6 z leading from the selection valvesare joined by a fluid coupling 203 which, depending on systemapplication or configuration, may comprise a mixing tee or a selectionvalve. If more than two selection valves 9 r are employed, then thecoupling 203 may comprise a multiport valve or a cross coupling. Thecoupling 203 may be configured so as to selectively fluidically coupleline 6 d to either one one of the pumps or to fluidically couple line 6d to both pumps simultaneously. Alternatively, the coupling 203 may beconfigured as a three-way tee valve which could accomplish eitherselective coupling (to one pump or the other) or simultaneous coupling(to both pumps). One-way check valves 201 a, 201 b may be installed inone or more of the fluid tubing lines 6 z so as to prevent fluidoriginating from a pump or valve in which the fluid is held at a highpressure from flowing back into a coupled second pump or valve in whicha fluid is maintained at a lower pressure. If it is known that one pumpwill always be operating at a higher pressure than other pumps, then acheck-valve may not be required on an output line associated with thatpump.

The sample-source sub-system 200 shown in FIG. 7A may be used in placeof the injector 5 shown in FIGS. 4A-4B. Accordingly, the sample-sourcesub-system 200 is illustrated, in FIG. 7A, as being fluidically coupledto the valve system 45 and associated two-column chromatographiccartridge 40 previously discussed in relation to FIG. 4B. Accordingly,an output fluid tubing line 6 f delivers a mixture of sample plussolvents to a port of valve v1 of the valve system 45. As previouslydiscussed, an output fluid tubing line 6 h from the chromatographiccartridge 40 delivers separated chemical components to a massspectrometer (not shown).

FIGS. 7B-7C are schematic illustrations of example solvent sourcesub-systems of an LCMS system according to some embodiments. Thesub-system 250 illustrated in FIG. 7B may be employed as an elutingsystem for separating analytes previously loaded onto a chromatographiccolumn. As shown, the solvent source sub-system 250 comprises many ofthe same components already described in relation to FIG. 7A. However,the sub-system 250 does not include an injector. Instead, solvents thatare either mixed or selected at the fluid coupling 203 are directed,along fluid tubing line 6 s, to a port of the valve v2 of the valvesystem 45. The sub-system 250 illustrated in FIG. 7C is similar to thesub-system 250 of FIG. 7C except that the sub-system 270 includes asecond sample injector apparatus 5 installed along the fluid tubing line6 s. The provision of the second sample injector apparatus gives usersthe ability to inject sample on the second injector and thereby bypassthe chromatographic column 7 a within the cartridge 40. At those timeswhen the second sample injector apparatus is not used, the valve portionof the second injector apparatus may be configured to route fluidreceived from the coupling 203 directly to valve v2. One-way checkvalves 201 d, 201 e may be installed in one or more of the fluid tubinglines leading from output ports of the pumps 9 r so as to prevent fluidoriginating from a pump or valve in which the fluid is held at a highpressure from flowing back into a coupled second pump or valve in whicha fluid is maintained at a lower pressure. If it is known that one pumpwill always be operating at a higher pressure than other pumps, then acheck-valve may not be required on an output line associated with thatpump.

Example 2—Observed Pressure Increase Versus Compressibility

In order to determine, during routine chromatograph operation, if ameasured pressure increase corresponds to a compressibility of anexpected fluid (e.g., Step 110 of Method 100 outlined in FIG. 6), it isdesirable to first generate pressure calibration data. FIG. 8 provides agraph 300 showing examples such data. The top two curves illustrated inFIG. 8—curve 302 and curve 304—are plots of pressure versus time for asobserved for bubble-free water and methanol, respectively, as each fluidis compressed against a plugged port by movement of a syringe pumppiston at a constant rate. In each case, compression is started at timet₁ and pressure is released at time t₂. As expected, the rate ofpressure increase in such experiments is inversely related to thecompressibility of each single-phase fluid within the syringe pump.However, if air or gas bubbles are present within a liquid, then therising-pressure portion of the curve will be delayed, as is indicated bycurve 308, which represents the same experiment performed on a two-phasefluid consisting of water and gas bubbles. If bubbles are present, theinitial motion of the pump piston serves to collapse gas bubbles and,perhaps, cause dissolution of the gas into the liquid withoutsubstantial pressure increase. Such results can thus be utilized tomonitor for and detect the presence of unwanted air or gas bubblesduring routine operation.

Example 3—Monitoring Pump Pressure Integrity and Lifetime

Fluid compression experiments may also be employed to determine thepressure integrity of seals with a syringe pump as well as to developpredictive models that can alert a user that a pump is approachingfailure. FIG. 9A schematically illustrates the general form of dataobtained in such experiments. The curve in graph 320 represents theobserved pump pressure during corresponding to two experimentalsegments. During the first such segment, a fluid is compressed byoperation of a syringe pump while an output valve is directed to aplugged port. During this experimental segment, the observed pressurerises along curve segment 321 a as a result of the finitecompressibility of the fluid. During the second experimental segment thesyringe pump is held motionless while the pressure drift—represented bycurve segment 321 b, is observed. During this time period, a slowdecrease in internal pump pressure is observed—even with a newpump—because the pump and valve pressure seals are necessarilyimperfect. Curve segment 321 c represents release of pressure at the endof an experiment.

The slope of the curve segment 321 b shown in FIG. 9A is diagnostic. Ifa syringe pump is repeatedly operated over many such compression anddecompression cycles, the pressure-sealing ability of the pump will beobserved to deteriorate over time. Stated differently, the leak rate,expressed as the rate of pressure change, will be observed to increaseover time. If experiments such as those described in relation to FIG. 9Aare periodically performed during the course of compression cycling,then the variation in leak rate versus pump lifetime may be modeled. Ifsuch experiments are then also periodically performed—eitherautomatically or under the control of a user—during the course ofroutine pump operation, then the observed leak rate behavior may becompared with the model in order to predict remaining pump lifetime.Since the exact rate of pressure decay (i.e., the leak rate) may varyboth with fluid type and with pump operating pressure, such leak rateexperiments should be performed under standardized conditions. Thepressure leak rate is observed to follow a curve such as the curve 352shown in graph 350 of FIG. 9B.

As indicated by the data plotted in FIG. 9B, the pressure leak rate of asyringe pump does not vary from its initial value, over an initialperiod encompassing most of the pump's usable lifetime, by more than acertain value. It is further observed that, at some time prior to totalfailure of the pump, the pressure-sealing ability of the pump will enterinto a pre-failure condition, at the onset of which the leak raterapidly increases to greater than twice the maximum leak rate observedduring the initial period. The onset of the pre-failure condition isthus indicated by the leak rate initially exceeding the normal workingthreshold line 351 as indicated in FIG. 9B.

During routine operation, a chromatograph instrument can periodically oroccasionally be operated so as to measure pressure decay according to aprocedure such as that discussed in reference to FIG. 9A. The pressuredecay measurements could be programmed to occur automatically at regularperiods. With such data, it is possible to predict how many injectioncycles may remain in a pump's useable lifetime according to model data(e.g., curve 352) previously determined. Once the pressure leak rate hasbeen observed to exceed such a threshold, a warning may be issued tousers that the pump is approaching the end of its useable lifetime andshould soon be repaired or replaced. Such a warning or alert may becommunicated during warning period w as indicated on FIG. 9B. Once theleak rate has exceeded a second threshold, indicated as warningthreshold line 353 in FIG. 9B, the operation of the pump may no longermeet specifications and the pump will soon undergo complete failure atregion f.

FIG. 13 is a flow diagram of a method for chromatography pump lifetimemonitoring and testing that illustrates the above concepts. In the firststep, step 452, of the method 470 (FIG. 13), a known solvent or reagentis drawn into a syringe pump. In general, a syringe pump comprises amechanically-driven piston which is fluidically sealed against and moveswithin a hollow cylinder so as to either draw fluid into a portion ofthe cylinder or to expel at least a portion of the fluid from thecylinder. If the output of the pump is blocked or if there is a blockagein a fluidic system to which the pump output is routed, then smallmovement of the piston will cause a compression of the fluid and a rapidpressure increase. Subsequent holding or maintaining of the syringe pumppiston in a fixed position should correspond to no pressure change, ifthe pressure seals of the pump are perfect. In practice, a small rate ofpressure decrease is normal and expected, since the seal is not perfect.With continued pump operation, however, wear in the seals and variousmechanical components may lead to increasing rates or pressure loss whenthe syringe pump piston is maintained in a fixed position. Additionally,if leaks are present, the rate of pressure increase may be less thanexpected upon movement of the piston so as to compress the fluid,provided that the compressibility of the fluid in the pump cylinder isknown.

Thus, in step 454 of the method 470 (FIG. 13), the fluid flow is routedto a blocked pathway. This step may be accomplished, for instance, byconfiguring a valve near the pump output—such as multiple-port rotaryselection valve 9 r illustrated in FIG. 3, so as to route the pumpoutput to a plugged port, such as, for instance port p1 shown in FIG. 2.Accordingly, the valve is used so as to fluidically couple the pump to aplugged port. In the next step, Step 456, the pump piston is caused tomove at a pre-determined rate in a direction which compresses the fluidbetween the piston and the plug. In Step 458, the compression is stoppedand the pump piston is maintained or held in a fixed position while thepressure decrease, preferably of the fluid within the pump, ismonitored. In the next step, Step 482, the rate of decrease is comparedto a standard profile, such as profile 352 shown in FIG. 9B, thatindicates the expected behavior of the pressure sealing capability of apump throughout its lifetime. The profile 352 may be pre-determined, forinstance, from prior experience with one or more pumps of the same typeas the one being tested. In Step 484, an estimated remaining usefullifetime of the pump is determined, based on the comparison made in Step482. If the estimated remaining lifetime (either in terms of time oroperational cycles) is less than a certain threshold (Step 486), then anotification is provided or an alarm is raised (Step 488), asappropriate.

Example 4—Monitoring Pump Precision

In general, the mechanical movement of a piston of a syringe pump iscontrolled by a lead screw that is mechanically coupled to the piston.Accordingly, the precision of the pump, as determined by the precisionof the fluid flow rate produced by action of the pump, depends upon theprecision of the thread pitch of the lead screw and the thread pitch ofmating threads in a mating threaded bore in which the lead screw moves.Likewise, deterioration in pump precision over time will be affected, atleast in part, by wear of the lead screw threads and mating threads.

If the pressure in a syringe pump chamber is continuously measuredduring operation of the pump so as to produce a constant nominal fluidflow rate through a flow resistive tubing (e.g., resistive tubing 6 c inFIG. 2) or other component with constant fluid flow resistance, then thepressure is observed to follow a pattern such as indicated in graph 400of FIG. 10. The two curves—curve 402 and curve 404—shown in FIG. 10represent pump chamber pressure measured during two separate operationsof a pump so as to pump water through a resistive tubing an a nominalconstant flow rate of 200 μL/min. The pressure variation so measured isobserved to vary cyclically with a periodicity that corresponds to thetime for the lead screw to undergo a single rotation. In regards to thedata plotted in FIG. 10, the average oscillatory pressure fluctuation isapproximately five-percent of the total pressure, which is found to benormal for a new pump. Since the flow rate will approach a uniformaverage value over several cycles, this oscillatory pressure profile maybe tolerable, depending upon the needs of users. However, someapplications may require a tight tolerance on flow rates andmeasurements such as those shown in FIG. 10 may be employed to determineif a particular pump is within tolerance. Further, for any pump, thetrend of ΔP may be monitored over the working lifetime of the pump, withany increase in this quantity being used to predict when wear on thepump mechanical parts will require pump maintenance or replacement.

Example 5—Detecting Leaks, Bubbles and Blockages in System

The pressure monitoring techniques described above in the context ofdetecting leaks of or air bubbles in syringe pumps may also be employedto detect problems relating to the fluidic components of an LSMS system.For instance, if there is a blockage in a fluid tubing line or othercomponent, then the observed pressure should be higher than expected fora normally operating clean system. On the other hand, leaks may bedetected by intentionally blocking or plugging one component of the LSMSsystem, pressurizing the portion of the fluidic system between the pumpand the intentional blockage and, then, monitoring for any unusuallyhigh decreases in pressure.

In order to identify a particular portion of an LCMS fluidic system thatis responsible for a problem, it is necessary to fluidically isolatespecific portions of the system. One means of accomplishing suchisolation is by replacing the two-column chromatographic cartridge 40(FIG. 4B, FIGS. 7A-7C) with a special test cartridge that is employedonly for system test purposes. Since the two-column chromatographiccartridge 40 is designed as a replaceable module, the replacementcartridge should be designed so as to be easily swapped for thecolumn-containing cartridge and to allow for easy re-insertion of thecolumn-containing cartridge.

FIG. 11A illustrates the two-column chromatographic cartridge 40 havinga first column 7 a and a second column 7 b. FIG. 11B shows a firsttest-related cartridge 40 b that is designed to be swappable with thecartridge 40. In the test-related cartridge 40 b, the columns 7 a, 7 bare replaced by two simple tubes 41 which are designed to permitunrestricted flow through the cartridge 40 b. By using the test-relatedcartridge 40 b, fluid can be routed through an entire LCMS system so asto detect any blockages. FIG. 11C illustrates a second test-relatedcartridge 40 c, which is related to the two-column cartridge 40 byreplacement of first column 7 a with a simple tube 41 that permitsunrestricted flow between fluid tubing lines 6 j and 6 k and replacementof the column 7 b by one or more plugs 42 so as to prevent flow betweenfluid tubing lines 6 q and 6 h. This configuration permits one portionof the fluidic system to be isolated for detection of leaks or bubbleswithin that portion while permitting free flow through the othersection. FIG. 11D illustrates a third test-related cartridge 40 d, whichis related to the two-column cartridge 40 by replacement of bothchromatographic columns 7 a, 7 b with plugs or other blockages 42.

The test-related cartridges illustrated in FIG. 11 are just a fewexamples. One can also easily envision an alternative cartridgeconfiguration, for instance, in which unrestricted fluid flow ispermitted between fluid tubing lines 6 q and 6 h while the couplingbetween fluid tubing lines 6 j and 6 k is plugged or otherwise blocked.One can also easily envision other cartridge configurations whichinclude a chromatographic column in one position with the other positionoccupied by a tube 41 or by one or more plugs 42.

FIG. 12A is a flow diagram of a general method for monitoring ordetection of leaks in a liquid chromatography system, in accordance withthe discussion in Example 5. The method 450 a illustrated in FIG. 12Amay be applied to leak detection and monitoring of pumps but, moregenerally, may also be applied to leak detection and monitoringthroughout an entire fluidic system. Steps 452-458 of the method 450 aare the same steps shown in and already discussed in regard to FIG. 13.However, it is here noted that the “blocked pathway” referred to in Step454 need not be limited to the vicinity of a pump but may be placedanywhere in the fluidic system. Thus, the blocked pathway may beassociated with any component anywhere in the system, such as a valve oran insertable and removable plug, or an insertable or removablecartridge, etc., which may be configured to inhibit flow past theblockage. Multiple such intentional blockages may be employed, insequence, at different points within the fluidic system so as to isolateand identify any leaks. In Step 456 of the method 450 a, the compressionof the fluid may occur not only in a pump but also within a portion ofthe fluidic system. Thus, some fluid may be necessarily expelled fromthe pump into the portion of the fluidic system. The pressure monitoringin Step 458 should preferably be performed with a pressure sensor inclose proximity to the fluidic system portion of interest. If, in thedecision step (Step 460) the pressure decrease determined in Step 458exceeds a certain pre-determined threshold, then an alarm is raised or anotification made in Step 462.

FIG. 12B is a flow diagram of a second method for monitoring ordetection of leaks in a liquid chromatography system in accordance withthe present teachings. The method 450 b illustrated in FIG. 12B issomewhat similar to the method 450 a (FIG. 12A) but includes additionalprovisions for monitoring multiple fluid pathways within an LC system,for detecting bubbles, for warning of impending pump failure and for atleast partially isolating the locations of leaks. The first steps—Step452 of drawing in a known solvent or other fluid and Step 454 of routingflow to an intentionally blocked pathway—of the method 450 b are thesame as the corresponding steps in the method 450 a. Then, in Step 455and Step 457, respectively, a fluid pathway is selected and the systemis configured—such as by configuring one or more valves—so as to routethe pump output to the selected fluid pathway. In Step 459, the fluid ispumped into the fluid pathway at a predetermined flow rate while thepressure is simultaneously measured.

In the decision step 461 of the method 450 b, if the pressure increasemeets the expected pressure increase profile—that is, if the pressureincrease is not less than that expected from a pre-determined profile,within tolerance—then pumping continues at Step 465. Otherwise (if thepressure increase is less than that expected), then one or more air orgas bubbles or pockets are interpreted to be present in the fluidpathway and a warning or notification of this condition is provided atStep 463. At this point, the method terminates (Step 464 a) so that auser or technician may prime the LC system, after which the method maybe started again from the beginning.

After a period of pumping fluid into the selected pathway (Step 465), adetermination is made (in Step 466) as to whether the selected fluidpathway is capable of achieving some pre-defined pressure within aprescribed time or within a prescribed movement of the pump piston. Ifnot, then a leak in either the pump plumbing system or the valve seal isinterpreted to be present and a warning or notification to this effectis made in Step 467. If the presumed leak is determined not to be inancillary pump plumbing components (Step 468) than a notification orwarning of a valve seal failure may be made in Step 469. At this point,the method terminates (Step 464 b) so that a user or technician may makeany necessary repairs and prime the LC system. After making such repairsand priming, the method may be started again from the beginning.

If it is determined, in Step 466, that the selected fluid pathway iscapable of achieving the pre-defined pressure within the prescribed timeor piston movement, then pump movement is stopped (Step 471) andpressure decrease is monitored for a certain pre-determined length oftime. If the change in pressure within the pre-determined time isdenoted as the negative quantity ΔP, then the pressure decrement—thatis, the amount by which the pressure decreases—is given as |ΔP|. Thispressure decrement is determined in Step 472 and, a subsequentdetermination is made in Step 473 as to whether this pressure decrementis less than a pre-defined normal working threshold. The normal workingthreshold is defined such that, if the pressure decrement is less thanthis threshold, then the fluidic components within the selected pathwayare presumed to be operating normally.

If the pressure decrement as defined above is greater than or equal tothe working threshold value, then a leak is presumed to be presenteither in fluidic plumbing components of the pathway or in a valve sealof a valve within the pathway. If, in Step 474, it isdetermined—possibly by visual inspection—that there are leaks in the LCplumbing system, then the method terminates at Step 464 b. Otherwise, ifthere are no leaks in the plumbing system, then a determination is made(Step 476) as to whether the pressure decrement is less than a warningthreshold. This determination may be considered to be a test of theseverity of any valve-seal leak. A pressure decrement above the normalworking threshold but below the warning threshold (the warning thresholdvalue being always greater than the normal working threshold value) isinterpreted to mean that a valve seal, while presently still useable, isin danger of failing in the near future. In such a situation, a warningto this effect is provided in Step 478, after which a different fluidpathway may be set in Step 479. If the pressure decrement is determined,in Step 476, to be greater than or equal to the warning threshold, thena valve-seal failure has occurred and a notification to this effect isprovided in Step 477 and the method terminates at Step 464 b.

Step 475 of the method 450 b (FIG. 12B) is executed if the pressuredecrement has been determined (Step 474) to be less than the normalworking threshold. In Step 475, the pump pressure is decreased to somepre-determined value, after which a different fluid pathway may beselected in Step 479 so that the method 450 b may be employed, from thebeginning, to test the different pathway.

Method 500 illustrated in flowchart form in FIG. 14 is a furthergeneralization of the previously discussed methods 480 (FIG. 13) and 450a (FIG. 12A). Thus, although Steps 452, 454 and 458 of the method 500are as previously described, the method includes a new step, Step 506,that replaces the previously described Step 456. In Step 506, pressureis monitored during the compression step so as to produce a firstsegment (segment #1) of a pressure profile. This is combined with thedecreasing pressure profile (segment #2) measured in Step 458 to yield amultiple-segment pressure profile. The measured multi-segment profile iscompared, in Step 508, to a multi-segment expected profile, such as theschematic profile illustrated in FIG. 9A. If the measured multi-segmentprofile is not the same as the expected profile, within a tolerance(Step 510), then an alarm is raised (Step 512). For example, bycomparing pressure changes during the compression step (i.e., segment#1) to expected values, air or gas bubbles within the general fluidicsystem may be detected. Such bubbles will be observed as an initialdelay in pressure increase, relative to expected profiles. In principle,multiple intentional blockages may be employed, in sequence, atdifferent points within the fluidic system so as to isolate and identifythe location of any included air or gas bubbles.

FIG. 15 illustrates another method for monitoring a liquidchromatography system, in accordance with the present teachings. Thefirst step, Step 452, of the method 550 (FIG. 15) is as previouslydescribed and comprises drawing a known solvent or reagent into a pump.In the next step, Step 554, the flow is routed through either anun-blocked fluidic pathway or through a fluidic pathway comprising aknown low resistance to fluid flow. In this context, an “un-blocked”pathway is a pathway within which an ordinarily-employed flow-resistivecomponent has been temporarily replaced, for testing or monitoringpurposes, by a simple tubing or otherwise open conduit, thereby enablingfree flow through the tubing or conduit and downstream portions of thefluidic pathway. For example, the replacement tubing or conduit mayreplace one or more chromatographic columns so as to permit free flow offluid through and past the nominal location of the column. The tubingsegments 41 in the special test-related cartridges 40 b and 40 c (FIGS.11B-11C) are examples of such temporarily installed open pieces oftubing. The fluid routing may be accomplished by means of configuring avalve, as has been previously described herein.

In Step 556 of the method 550 (FIG. 15), a particular fluid pathway isselected. Then in Step 558, various valves may be configured to routethe solvent, reagent or other fluid to the selected pathway. In Step559, the fluid is at least partially discharged from the pump andthereby caused to flow using a pre-defined or a real-time-calculatedflow rate through the low resistance element or un-blocked pathway ofthe fluidic system (as provided in Step 554), during which fluidpressure is monitored in real-time. The accumulated fluid pressure datacomprises a measured pressure profile, which is compared to an expectedpressure profile in Step 561. Alternatively, one or more individualpressure data points (at certain times) may be employed, instead of afull profile. If the measured profile (or value of a measured pressuredata point) is greater than, within a tolerance, the expected orspecified profile or value (Step 561), then an alarm is raised or othernotification provided (Step 562) that flow through at least a portion ofthe system is blocked. The entire procedure may be repeated with adifferent selected pathway (Step 563). The expected profile may dependupon the particular fluid properties of the known solvent. The entirepressure profile or any portion of the pressure profile may be employedfor making the comparison in the decision step 561.

Example 6—Real-Time Pressure Compensation

Using the LCMS system configurations illustrated in FIGS. 7A-7C, it ispossible for pressure imbalances to develop between different sectionsof an overall system. Real-time pressure monitoring and compensation maybe employed to re-balance the pressures. At least two differentscenarios, described below, are possible.

In a first pressure-compensation scenario, two initially mutuallyisolated portions of a fluidic system are initially pressurized atdifferent respective pressures. The different pressures could arise assimply as a consequence of one pump—associated with the firstportion—being inactive at the time that a second pump—associated withthe second portion—is operating. The different pressures could alsovariously arise as a consequence of different fluid properties,different required flow rates or different inherent flow resistances inthe two portions. In this scenario, the two portions are subsequentlyfluidically coupled for the purpose of blending or mixing of the fluids.The fluidic coupling between the two system portions may be broughtabout by re-configuring a valve, for instance. When the two portions arefluidically interconnected, conventional systems will experience asudden pressure drop in the portion that was initially at the higherpressure.

To prevent an unwanted pressure drop from occurring in the scenariodescribed above, the following steps, according to the instant teachingsand outlined in FIG. 16A, may be employed: (a) directing the output ofthe pump associated with the lower-pressure portion (fluid line #1, foridentification purposes) to a plugged output (Step 602 of method 600shown in FIG. 16A); (b) compressing the fluid within the pump whilemonitoring the pressure of the higher pressure portion (fluid line #2),thereby matching the pressure in the pump to the pressure in fluid line#2 (Step 604); directing the output of the pump to the fluid line #1 soat to increase the pressure within fluid line #1 (Step 606); andfluidically interconnecting fluid line #1 and fluid line #2 (Step 608).Step 602 of directing the pump output to a plugged output may beperformed, for example, by rotating an appropriate rotary valve 9 r(FIGS. 2, 7) to one of the plug positions shown by cross-hatching, suchas plug position p1 illustrated in FIG. 2.

FIG. 16B is a flow diagram of a method for compensating pressuredifferences in two fluid lines which are to be employed for fluid flowat flow rates specified by a particular liquid chromatography method. InStep 652, the pressures of fluid line #1 and fluid line #2 are monitoredor measured. Then, in Step 654, the two fluid lines are interconnected,such as by configuring one or more interconnection valves. In Step 656,an expected final equilibrium pressure (or pressures) is (are)calculated based on the pressures before interconnection and as afunction of flow rate. In Step 658, the target pressures of the pumpsare set at the expected equilibrium pressure or respective pressures by,for example, compressing fluid within the pumps against plugged outputports. Then, in Step 659, the flow rates are controlled so as to reachthe target pressure using a control algorithm such as the well-knownproportional-integral-derivative (PID) control algorithm. Finally, inStep 661, the flow rates are rates are re-set to the specific flow ratesspecified in a particular liquid chromatographic method that is to beperformed by the system.

In another pressure-compensation scenario, pressure compensation may beemployed so as to balance pressure differences between a loading pumpsub-system, perhaps configured similar to sub-system 200 shown in FIG.7A and an eluting pump sub-system, possibly configured similar tosub-system 250 or sub-system 270 illustrated in FIG. 7B and FIG. 7C,respectively. For example, if column 7 a (FIGS. 4, 5 and 7) is aTurboFlow® column as described above and column 7 b is an analyticalcolumn, then the fluid directed from the loading pump system into valvev1 and column 7 a will generally be at a nominal operating pressure of3-7 MPa and the fluid directed from the eluting pump into valve v2 willgenerally be at a nominal operating pressure of 30-40 MPa.

In many systems, and as is shown in middle diagram of FIG. 5, the rotaryvalve v2 of valve system 45 has an internal structure which enablesthree adjacent ports to be fluidically simultaneously interconnected.This valve structure enables, for instance, separate fluid flows fromfluid tubing lines 6 n and 6 s, respectively from the loading andeluting sub-systems, to be mixed in valve v2 and output, as a mixture,to fluid tubing line 6 q. In order to prevent a sudden pressure drop andconsequent incorrect flow of fluid when the elution begins, a checkvalve (not shown) may be provided in the fluidic system between anoperative loading pump and the point at which the fluid pathways fromthe loading pump sub-system and eluting pump sub-system converge. Thecheck valve will not operate so as to permit flow from the loading pumpsystem until the pressure is equalized. Then, with valve v2 configuredfor elution, the pressure applied by the loading pump is ramped higheras fast as possible until equal pressures exist on both sides of thecheck valve.

Example 7—Pre-Compression and Pre-Pressurization

Using the LCMS system configurations illustrated in FIGS. 7A-7C, it isoccasionally necessary to execute a pre-compression step—prior todirecting fluid flow through a chromatographic column—so as to ramp thefluid pressure up to a nominal operating pressure for applicationsemploying the column. In the case of loading analytes onto a TurboFlow®column (described above), the nominal operating pressure and flow rateare generally specified by a user as part of a chromatographic method.However, because of the behavior of the column, some flow-through timeis required for the fluid in the column to come up to the correctpressure. As a result of this behavior, the initial flow of sample intothe TurboFlow® column may not occur at the user-specified pressure.Accordingly, prior to performing the first step (Step 1) of the usermethod, the pressure of a pump that is used to load the column may firstbe directed to a plugged port. With the pump output so configured, thefluid in the pump compressed so as to initially ramp up the pumppressure, prior to directing the sample into the column. Performing thispre-pressurization step allows the entire sample loading step (or otherflow-through step, depending on the type of column) to be performed atthe correct pressure.

Example 8—Combined Fluid Monitoring and Pump Diagnostics

The method 700 illustrated in flowchart form in FIG. 17 combines bothfluid monitoring and generation of pump diagnostic information. In thefirst step, step 702, of the method 700 (FIG. 17), a known solvent orreagent is drawn into a syringe pump. In step 704, the fluid flow isrouted to a plugged output port such as, for instance, a port ofmultiple-port rotary selection valve that is in proximity to the pump.In Step 706, the pump piston is caused to move at a pre-determined ratein a direction which compresses the fluid between the piston and theplug.

In the decision step 708 of the method 700, if the pressure increasemeets the expected pressure increase profile—that is, if the pressureincrease is not less than that expected from a pre-determined profile,within a tolerance—then pumping continues at Step 714. Otherwise (if thepressure increase is less than that expected), then one or more air orgas bubbles or pockets are interpreted to be present in the fluidpathway and a warning or notification of this condition is provided atStep 710. At this point, the method terminates (Step 712) so that a useror technician may prime the LC system, after which the method may bestarted again from the beginning.

After a period of continued compression (Step 714), a determination ismade (in Step 716) as to whether the pump is capable of achieving somepre-defined pressure. If not, then a leak in either the pump plumbingsystem or the or the pump (e.g., valve seal or pistion) is interpretedto be present and a warning or notification to this effect is made inStep 718. If the presumed leak is determined not to be in ancillary pumpplumbing components (Step 720) than a notification or warning of a pumppiston or valve seal failure may be made in Step 722. At this point, themethod terminates (Step 724) so that a user or technician may make anynecessary repairs and prime the LC system. After making such repairs andpriming, the method may be started again from the beginning.

If the pre-defined pressure has been attained, then solventcompressibility is calculated in 726 using the amount of piston movementrequired to achieve the pre-defined pressure. If the compressibility isnot as expected for a presumed solvent or other fluid, then awrong-solvent warning is provided in step 730 and the method terminates.If, however, the compressibility is determined to be as expected, withina tolerance, then pump movement is stopped (Step 732) and pressuredecrease is monitored for a certain pre-determined length of time. Thepressure decrement, |ΔP|, is determined in Step 734 and, a subsequentdetermination is made in Step 736 as to whether this pressure decrementis less than a pre-defined normal working threshold. The normal workingthreshold is defined such that, if the pressure decrement is less thanthis threshold, then the pump and any associated components are presumedto be operating normally. If the pressure decrement as calculated inStep 734 is greater than or equal to the working threshold value, then aleak is presumed to be present either in the pump or associatedcomponents. If the pressure decrement is further determined, in Step738, to be greater than or equal to a warning threshold, then a failurehas occurred in either the pump or an associated component and anotification to this effect is provided in Step 742. (Note that thewarning threshold value is always greater than the normal workingthreshold value.) After the notification provided in Step 742, themethod terminates at Step 744, so that the pump may be repaired orreplaced and the system re-primed.

A pressure decrement above the normal working threshold as determined inStep 736 but below the warning threshold as determined in Step 738 isinterpreted to mean that a pump component, such as a piston, or arelated component, such as a valve seal, while presently still useable,is in danger of failing in the near future. In such a situation, awarning to this effect is provided in Step 740, after which Step 746 isentered.

Step 746 is executed if the pressure decrement is determined to be belowthe warning threshold. In Step 746, the pressure is decreased to acertain pre-determined value. Then, in Step 748, the flow is routed to apathway having a constant fluid flow resistance, and caused to flow at apre-determined flow rate in Step 750 while pressure is monitored. InStep 752, the viscosity of the fluid is calculated using an averagemonitored pressure. If it is determined (Step 760) that the fluidviscosity is not as expected for a presumed fluid, then a wrong-solventwarning or notification is provided in Step 762 and the methodterminated. However, if the calculated viscosity is as expected, withina tolerance, then a pressure fluctuation, versus time or pistonmovement, is determined (in Step 754) using the pressure variation thatwas monitored in Step 750. For example, the pressure fluctuation couldbe similar to the periodic curves 402 and 404 shown in FIG. 10. In thissituation, a range of the fluctuation could be determined as the averageor possibly maximum peak-to-peak pressure variation or as a standarddeviation of the fluctuation or could be determined in some otherfashion. Regardless of the method by which the range is characterized, arange greater than a certain pre-determined range threshold, asdetermined in Step 756, is taken as an indication of excessivemechanical wear in moveable pump parts. Accordingly, a fluctuation rangegreater than the threshold (Step 756) will cause a mechanical-wearwarning or notification to be provided in Step 758, after which themethod terminates at Step 744, so that the pump may be repaired orreplaced and the system re-primed.

All of the pump-diagnostic methods and system-diagnostic methodsdescribed in certain of the examples given above may be performed atcertain dedicated system-test times when no chromatographic separationsof samples are being run. However, since many liquid chromatographymethods necessarily require pre-compression and pressure ramping, thesystem monitoring can be built in and can occur automatically whenrunning the various user-specified liquid chromatography methods.

An improved liquid chromatography system has been disclosed.Advantageously, a system in accordance with the present teachings may beemployed in an automated sample preparation and analysis system, such asis disclosed in a co-pending International (PCT) application for patenttitled “Automated System for Sample Preparation and Analysis” (AttorneyDocket No. TFS-13AWO, Application No. PCT/US11/58452) filed on Oct. 28,2011 and incorporated herein by reference in its entirety. In variousembodiments, the automated sample preparation and analysis systemincludes a sample preparation system for preparing various samples and asample analysis system, which may include a liquid chromatography massspectrometer (“LCMS”) for analyzing the prepared samples according toselected analyte assays. The sample preparation system and the sampleanalysis system are interconnected in an automated manner. The automatedsample preparation and analysis system is designed to generally operatewith minimal operator intervention or maintenance and includes at leastone controller for, inter alia, controlling valve configurations and,optionally, monitoring operational or instrumental conditions. Becauseof the automated nature of the instrument, it is advantageous for theautomated system to be able to monitor its own configuration andoperating state and to provide an alert an operator if the systemdetects any possible problems. A system for liquid chromatography inaccordance with the present teachings may assist in these functions.

The discussion included in this application is intended to serve as abasic description. Although the present invention has been described inaccordance with the various embodiments shown and described, one ofordinary skill in the art will readily recognize that there could bevariations to the embodiments and those variations would be within thespirit and scope of the present invention. The reader should be awarethat the specific discussion may not explicitly describe all embodimentspossible; many alternatives are implicit. For example, it is easy toenvisage that various sub-sets of steps provided in flowcharts hereinmay be combined with sub-sets of steps from different flowcharts toarrive at augmented or hybridized methods. Accordingly, manymodifications may be made by one of ordinary skill in the art withoutdeparting from the spirit, scope and essence of the invention. Neitherthe description nor the terminology is intended to limit the scope ofthe invention. All patent application disclosures, patent applicationpublications or other publications are hereby explicitly incorporated byreference herein as if fully set forth herein.

What is claimed is:
 1. A system for monitoring a liquid chromatographysystem comprising: a valve; a container having a known fluid therein; asyringe pump having a piston, wherein the syringe pump is fluidicallycoupled to the valve; a pressure gauge sensor configured to measure apressure within the syringe pump; and a computer or electroniccontroller electrically or electronically coupled to the valve, thesyringe pump and the pressure gauge or sensor, the computer orelectronic controller comprising computer-readable instructions that areoperable to: cause the syringe pump to draw the fluid from the containerinto the syringe pump; configure the valve so such that the syringe pumpis fluidically coupled to either a fluidic pathway through the fluidicsystem or to a plug that prevents fluid flow; cause the piston of thesyringe pump to move at a predetermined rate in a direction so as toprogressively compress the fluid therein or expel the fluid to thefluidic pathway, while reading a pressure of the fluid measured by thepressure gauge or sensor; determine a profile of the variation of themeasured pressure for the time that the piston is caused to move;compare the determined profile to an expected profile that depends uponthe fluid; and provide a notification of a sub-optimal operatingcondition or malfunction if the determined profile varies from theexpected profile by greater than a predetermined tolerance.
 2. A systemas recited in claim 1, wherein the computer-readable instructions thatare operable to configure the valve are operable to configure the valvesuch that the syringe pump is fluidically coupled to a fluidic pathwayhaving an intentional flow blockage therein that prevents flow throughthe fluidic system beyond the intentional flow blockage.
 3. A system asrecited in claim 2, wherein the intentional flow blockage is provided ata nominal position of a chromatographic column within the fluidicsystem.
 4. A system as recited in claim 2, wherein the intentional flowblockage is provided in a cartridge that is disposed within the fluidicsystem at a nominal position of a two-column-bearing cartridge and thatis interchangeable with the two-column-bearing cartridge.
 5. A system asrecited in claim 1, wherein the computer-readable instructions that areoperable to provide a notification are operable to provide anotification that an air or gas bubble is present within the fluidicsystem if the determined pressure profile includes a delay in anincrease in pressure, relative to the expected profile.
 6. A system asrecited in claim 1, wherein the computer-readable instructions that areoperable to configure the valve are operable to configure the valve suchthat the syringe pump is fluidically coupled to a fluidic pathway havinga known resistance to fluid flow, and wherein the computer-readableinstructions that are operable to compare the determined profile to anexpected profile are operable to compare the determined profile to anoscillatory profile, the oscillations of said profile relating tomechanical movement within the syringe pump.
 7. A system as recited inclaim 1, further wherein the computer-readable instructions are furtheroperable to: cause the piston of the syringe pump to remain in a fixedposition, while reading a pressure of the fluid measured by the pressuregauge or sensor; determine a rate of decrease of the measured pressurewhile the piston is in the fixed position; compare the determined rateof pressure decrease to a model relating rate of pressure decrease toremaining pump lifetime; and provide a prediction of remaining pumplifetime based on the comparing.
 8. A system as recited in claim 1,wherein the computer-readable instructions are further operable to:cause the piston of the syringe pump to remain in a fixed position,while reading a pressure of the fluid measured by the pressure gauge orsensor; determine a rate of decrease of the measured pressure while thepiston is in the fixed position; compare the determined rate of pressuredecrease to a model relating rate of pressure decrease to remaining pumplifetime; and provide a warning that the syringe pump should be replacedor serviced based on the comparing.
 9. A system as recited in claim 1,wherein the computer-readable instructions that are operable toconfigure the valve are operable to configure the valve such that thesyringe pump is fluidically coupled to a fluidic pathway that includesthe nominal position of a chromatographic column, wherein the column isreplaced by a length of empty tubing, and wherein the computer-readableinstructions that are operable to provide a notification are operable toprovide a notification that the fluid pathway is blocked if thedetermined pressure profile includes a pressure increase that exceeds anexpected increase in pressure by greater than the predeterminedtolerance.
 10. A system as recited in claim 9, wherein the length ofempty tubing is provided in a cartridge that is disposed within thefluidic system at the nominal position of a two-column-bearing cartridgeand that is interchangeable with the two-column-bearing cartridge.